Hepatitis E Virus (HEV) was firstly identified as a pathogen of enterically transmitted non-A, non-B hepatitis in 1983 (Balayan et al., 1983. Intervirology 20:23). Hepatitis E is endemic mainly in developing countries in Asia, Africa and Middle America. In developed countries, hepatitis E cases were mostly found in immigrants or traveler from abroad. Both sporadic cases and pandemic have been reported. During the period from 1950s to 1990s, several hepatitis E outbreaks happened due to polluted drinking water (Visvanathan, 1957, Indian J. Med. Res. (Suppl.). 45:1-30; Wong et al., 1980 Lancet., 2:882-885; Myint et al., 1985, Am J Trop Med Hyg., 34:1183-1189; Belabbes et al., 1985 J Med Virol., 16:257-263; Hau et al., 1999, Am J Trop Med Hyg., 60:277-280). Most hepatitis E infection was self-limited and scarcely developed into a chronic disease; but for the pregnant women, the sequel was severe with a mortality rate up to above 17% (Tsega et al., 1992, Clin. Infec Dis., 14:961-965; Dilawari et al., 1994, Indian J Gastroenterol., 13:44-48; Hussaini et al., 1997, J Viral Hepat., 4:51-54).
In 1991, researchers got the complete genome sequence of HEV for the first time, and it was found that HEV is a single-strand non-enveloped positive RNA virus (Tam et al., 1991, Virology 185:120-131). Sequence analysis showed the genome was about 7.2 kb long with three open reading frames. ORF1, which is located at 5′ end, encodes non-structural protein of the virus, and ORF2, which is located at 3′ end, encodes major structural proteins of the virus. At 5′ end of ORF3, there is one base overlapped with ORF1 3′ end. At 3′ end of ORF3, there are 339 bases overlapped with ORF2. It's acknowledged that ORF3 encodes another structure protein with unknown function (Tam et al., 1991, Virology, 185:120-131; Aye et al., 1992, Nucleic Acids Res., 20:3512; Aye et al., 1993, Virus Genes., 7:95-109; Huang et al., 1992, Virology, 191:550-558; Reyes et al., 1993, Arch Virol Suppl., 7:15-25).
HEV ORF2, beginning at the base no. 5147, has 1980 nucleotides, which encodes a polypeptide with 660 amino acids presumed to be the major structural protein constituting the capsid of virus. At N-terminal of ORF2 protein, there is a classical signal peptide sequence followed by an arginine-rich region, which is highly positive charged region and is believed to involve in genomic RNA encapisidation during virus assembly. During the translation process, ORF2 enters endoplasmic reticulum (ER) by a mechanism of signal peptide recognizing protein (SRP), and is glycosylated and accumulated in ER, then probably forms the capsomer of capsid in suit. Three N-glycosylated sites on ORF2, Asn-137, Asn-310 and Asn-562, are highly conservative among different virus strains, and Asn-310 is the major glycosylated site. ORF2-transfected mammalian cells COS and human hepatocarcinoma cells Huh-7 and HepG2 can thereby express a 88 kD glycoprotein which can be found in both cytoplasma and membrane. The mutation in those glycosylated sites did not affect the location of PORF2 onto cell membrane. However after the signal peptide sequence was removed therefrom, PORF2 can only be found in cytoplasma. This implied that the shift of PORF2 instead of glocosylation is necessary for protein location onto cell membrane. Like MS protein in HBV, PORF2 is possibly secreted to cell membrane directly through ER, not through Golgi body. On the surface of transfected cell, gpORF2 is not randomly distributed, but concentrated in some zone, which implied an active combination process of protein subunits, which maybe aggregate into certain more ordered advanced forms. The final assembly/maturation of the virus need the encapisidation of genomic RNA, thus must occur in cytoplasma outside of ER or inner wall of cell membrane. The accumulation of gpORF2 on membrane may imply the assembly of virus. At the same time, the localization of capsid protein on membrane also implied the possibility of secretion of matured virus from the cell through budding.
In U.S. Pat. No. 5,885,768, Reyes et al. firstly reported that 4 cynomolgus monkeys were injected i.m. at days 0 and 30 with recombinant protein trpE-C2 expressed in E. coli comprising HEV Burma strain ORF2 C terminal 2/3 (aa225˜660) in an amount of 50 μg/dose, wherein said protein is formulated with an alum adjuvant. Two monkeys as controls were injected with adjuvant only. In bloods collected four weeks later no antibody is detected by Western Blotting. A third-time immunization on two monkeys among them by administering 80 μg per animal unsolvable recombinant protein without adjuvant. Four weeks later, both monkeys were positive (WB). Then the six monkeys were grouped into two groups, each including three monkeys: one immunized three-times, one two-times, and one as control. The first group was attacked with Burma HEV, and the second group Mexico HEV. The results were that (1) ALT was normal all the time in the immunized group, but it increased 6˜10 times higher than before immunization in control; (2) when liver biopsy sample was detected by Immunological Fluorescence method, the antigen was detected in all other monkeys except those immunized with three doses and attacked by Burma strain; (3) virus excretion in feces was found in all other monkeys except those immunized with three doses and attacked by Burma strain. This research sample was small, but it implied that recombinant protein from ORF2 could block the occurrence of biochemical indexes of virus hepatitis and protect completely some monkeys from infection when the monkeys were attacked by wild HEV.
Anderson group in Australia (Anderson et al., 1999. J. Virol. Methods., 81:131-142; Li et al., 1994, J Clin Microbio. 32:2060-2066; Li et al., 1997 J. Med. Virol., 52:289-300; Li et al., 2000, J. Med. Virol., 60:379-386) used ORF2 aa394˜660(ORF2.1) expressed in E. coli. The expression product was a fusion protein with GST or poly-His which could form a highly conformation-dependent convalescence epitope. This epitope could detect a high rate of convalescence sera, but it disappeared when the fragment was extended towards N-terminal or truncated. The serum 30 weeks after mice were immunized with recombinant ORF2.1 protein was used to block the serum from convalescence patients with VLP expressed in baculovirus as the coated antigen. The blocking rate reached 81%˜86%. Different data showed that ORF2.1 had a major epitope structure rather similar to VLP. The antibody to the epitope can exist for a long time in serum of HEV infected individuals. It's probably an important protective epitope, and antibodies to ORF2.1 would be of value.
In order to overcome many problems during the process of preparation and application of HEV monoclonal antibody, on the basis of obtaining a series of polypeptide fragments with excellent antigenecity within hepatitis E virus ORF2 (see Chinese patent No. CN00130634.0), the inventors prepared monoclonal antibody with NE2 fragment as antigen by hybridoma technology and obtained the cell line which can secrete the monoclonal antibody specifically binding the polypeptide encoded by hepatitis E virus ORF2 and the monoclonal antibody produced by said cell line.